Nitric oxide production in human endothelial cells stimulated by histamine requires Ca2+ influx.
The causal relationships between cytosolic free-Ca2+ concentration ([Ca2+]i) increases and production of nitric oxide (NO) have been investigated mostly with indirect methods and remain unclear. Here we demonstrate, by direct real-time measurements of [NO] with a porphyrinic microsensor, that Ca2+ entry, but not an increase in [Ca2+]i, is required for triggering of NO production in human endothelial cells. Histamine, ranging from 0.1 to 100 microM, increased both NO production and [Ca2+]i when given in a single dose. However, histamine caused increased NO release but induced progressively smaller [Ca2+]i changes when cumulatively added. In the absence of a transmembrane Ca2+ gradient, no significant NO release was detectable, despite the marked Ca2+ peak induced by histamine. Inhibition of Ca2+ entry by SK&F 96365 abolished histamine-elicited NO production but only reduced the transient [Ca2+]i rise. The suppression of the sustained [Ca2+]i response under these two conditions suggests that NO release was closely associated with Ca2+ entry from the extracellular space. In addition, membrane depolarization, achieved by increasing the extracellular K+ concentration from 5 to 130 mM, reduced both the amplitude of histamine-induced sustained [Ca2+]i elevation and NO production. These results lead us to propose that the availability of numerous Ca2+ ions around the internal side of the plasma membrane would promote the association between nitric oxide synthase and calmodulin, thereby activating the enzyme.
Lantoine
F
F
Pharmacology, URA CNRS 1482, Paris V University, Necker Medical School, 156 rue de Vaugirard, 75015, Paris, France.
Iouzalen
L
L
Devynck
M A
MA
Millanvoye-Van Brussel
E
E
David-Dufilho
M
M
D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D011188:Potassium / Q000378:metabolism
D002121:Calcium Channel Blockers; D007093:Imidazoles; D009569:Nitric Oxide; D006632:Histamine; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D011188:Potassium; D002118:Calcium